Recombinant cells that highly express chromosomally-integrated heterologous genes

ABSTRACT

Recombinant host cells are obtained that comprise (A) a heterologous, polypeptide-encoding polynucleotide segment, stably integrated into a chromosome, which is under transcriptional control of an endogenous promoter and (B) a mutation that effects increased expression of the heterologous segment, resulting in enhanced production by the host cells of each polypeptide encoded by that segment, relative to production of each polypeptide by the host cells in the absence of the mutation. The increased expression thus achieved is retained in the absence of conditions that select for cells displaying such increased expression. When the integrated segment comprises, for example, ethanol-production genes from an efficient ethanol producer like  Zymomonas mobilis , recombinant  Escherichia coli  and other enteric bacterial cells within the present invention are capable of converting a wide range of biomass-derived sugars efficiently to ethanol.

This application is a continuation-in-part of co-pending applicationSer. No. 07/352,062 (filed May 15, 1989), which is acontinuation-in-part of application Ser. No. 07/239,099 (filed Aug. 31,1988), now abandoned.

Work relating to this invention was supported in part by GrantFG05-86ER3574 from the Office of Basic Energy Science, U.S. Departmentof Energy, and in part by Grant 88-37233-3987 from the Alcohol FuelsProgram, U.S. Department of Agriculture. The U.S. Government has certainrights in the invention.

BACKGROUND OF THE INVENTION

The present invention relates to recombinant host cells that comprise aheterologous, polypeptide-encoding polynucleotide segment which isstably integrated into a chromosome and which is under control of anendogenous promoter. When the integrated segment comprises, for example,ethanol-production genes from an efficient ethanol producer likeZymomonas mobilis, recombinant Escherichia coli and otherenterobacterial cells within the present invention are capable ofconverting a wide range of biomass-derived sugars efficiently toethanol. This invention also relates to mutations that enhanceproduction of proteins encoded by chromosomally-integrated, heterologousgenes which are expressed under the control of an endogenous promoter,and to methods of identifying such mutations.

During glycolysis, cells convert simple sugars, such as glucose, intopyruvic acid, with a net production of ATP and NADH. In the absence of afunctioning electron transport system for oxidative phosphorylation, atleast 95% of the pyruvic acid is consumed in short pathways whichregenerate NAD⁺, an obligate requirement for continued glycolysis andATP production. The waste products of these NAD⁺ regeneration systemsare commonly referred to as fermentation products.

Microorganisms are particularly diverse in the array of fermentationproducts which are specific for each genus. See, for example, Krieg, N.R., and J. G. Holt, eds. [1984] BERGEY'S MANUAL OF SYSTEMATICBACTERIOLOGY (Williams & Wilkins Co., Baltimore). These products includeorganic acids, such as lactate, acetate, succinate, and butyrate, aswell as neutral products like ethanol, butanol, acetone, and butanediol.Indeed, the diversity of fermentation products from bacteria has led totheir use as a primary determinant in taxonomy. Krieg and Holt [1984],supra.

End products of fermentation share several fundamental features. Theyare relatively nontoxic under the conditions in which they are initiallyproduced but become more toxic upon accumulation. They are more reducedthan pyruvate because their immediate precursors have served as terminalelectron acceptors during glycolysis. The microbial production of thesefermentation products forms the basis for our traditional and mosteconomically successful applications of biotechnology and includes dairyproducts, meats, beverages, and fuels.

Most fuel ethanol is currently produced from hexose sugars in cornstarch or cane syrup utilizing either Saccharomyces cerevisiae orZymomonas mobilis (Z. mobilis). However, these are relatively expensivesources of biomass sugars and have competing value as foods. Inaddition, during fermentation much of the hexose is necessarilyconverted back to biomass, comprising microbial cells, rather than toethanol. For conventional ethanol-producing microorganisms, this biomasshas limited commercial value at best, for instance, as a nutritionalsupplement, and therefore, represents inefficient utilization of theexpensive sugar substrate.

Starches and hexose sugars represent only a fraction of the totalcarbohydrates in plants. The dominant forms of plant carbohydrate instems, leaves, hulls, husks, cobs, etc., are the structural wallpolymers, cellulose and hemicellulose. Hydrolysis of these polymersreleases a mixture of neutral sugars which include glucose, xylose,mannose, galactose, and arabinose. No known organism in nature canrapidly and efficiently metabolize all of these sugars, particularly thepentoses, into ethanol or any other single product of value.

Escherichia coli (E. coli) and related enteric bacteria are the maincommercially useful microorganisms that are capable of metabolizing theentire range of biomass-derived sugars by fermentation under anaerobicconditions. However, under anaerobic fermentation conditions, theseorganisms convert sugars to a mixture of soluble products, includingsmall amounts of ethanol, that cannot be separated economically. SeeIngram, L. O., T. Conway, D. P. Clark, G. W. Sewell, and J. F. Preston[1987] Appl. Environ. Microbiol. 53: 2420-2425. Thus, such entericbacteria efficiently utilize the entire range of biomass-derived sugarsbut fail to produce a product of sufficient yield and uniformity to becommercially valuable.

Accordingly, there is a need for microorganisms which combine theefficient metabolism of the entire range of biomass-derived sugars,which is exhibited by certain enteric bacteria, such as E. coli, withthe ability to produce high levels of a single, predominant, solublefermentation product of commercial value, such as ethanol. Further,there is a continuing need for such organisms that can produce microbialbiomass comprising additional products, such as commercially valuableproteins, in sufficient yield and quality for economical recovery.

Fermentation pathways transform pyruvic acid into a mixture of acidicand neutral products. Two pathways dominate in enteric bacteria such asE. coli. Lactate dehydrogenase catalyzes the reduction of pyruvate tolactic acid, directly oxidizing NADH to NAD⁺. The second pathway,involving pyruvate formate-lyase, is more complicated. Pyruvateformate-lyase, which catalyzes the cleavage of pyruvate to formate plusacetyl-coenzyme A, is a central enzyme of the anaerobic metabolism of E.coli, because under anaerobiosis this enzyme is responsible formetabolizing a large fraction of pyruvate. The E. coli gene encodingpyruvate formate-lyase (pfl gene) has been cloned and sequenced. SeeChristiansen, L., and S. Pedersen [1981] Mol. Gen. Genet. 181: 548-551;Rodel, W., W. Plaga, R. Frank, and J. Knappe [1988] Eur. J. Biochem.177: 153-158. The pfl gene is preceded by multiple promoters, and it isinduced to high levels of expression by anaerobiosis. See Sawers, G.,and A. Bock [1988] J. Bacteriol. 170: 5330-5336.

The DNA used to provide ethanol-production genes for a recombinant hostof the subject invention is isolated, for example, from Z. mobilis. Thisis a microorganism with unusual metabolic characteristics which iscommonly found in plant saps and in honey. Wild-type Z. mobilis has longserved as a natural inoculum for the fermentation of the Agave sap toproduce pulque, a Mexican alcoholic beverage, and as an inoculum forpalm wines. As noted above, this organism is also used for fuel ethanolproduction and has been reported to be capable of ethanol productionrates which are substantially higher than those of yeasts.

Although Z. mobilis is nutritionally simple and capable of synthesizingamino acids, nucleotides and vitamins, the range of sugars metabolizedby this organism is very limited and normally consists of glucose,fructose and sucrose. Substrate level phosphorylation from thefermentation of these sugars is the sole source of energy forbiosynthesis and homeostasis. Z. mobilis is incapable of growth withouta fermentable sugar even in rich medium such as nutrient broth.

In Z. mobilis, two enzymes, pyruvate decarboxylase (PDC) and alcoholdehydrogenase, particularly form II (ADHII), are required to convertpyruvate to ethanol and regenerate NAD⁺. High levels of the individualproteins are found in the cytoplasm of Z. mobilis, ranging from 2% to 5%each of the soluble protein. Such high levels are presumed to beessential for the high rates of NADH oxidation and glycolytic fluxrequired for energy production. The cloning and sequencing of Z. mobilispdc and adhB genes encoding PDC and ADHII, has been previously reported.See Conway, T., Y. A. Osman, J. I. Konnan, E. M. Hoffman, and L. O.Ingram [1987]J. Bacteriol. 169: 949-954; Conway, T., G. W. Sewell, Y. A.Osman, and L. O. Ingram [1987] J. Bacteriol. 169: 2591-2597; Brau, B.,and H. Sahm [1986] Arch. Microbiol. 146: 105-110; Brau, B., and H. Sahm[1986] Arch. Microbiol. 144: 296-301; Neale, A. D., R. K. Scopes, R. E.H. Wettenhall, and N. J. Hoogenraad [1987] Nucleic Acid. Res. 15:1753-1761; Ingram, L. O., and T. Conway [1988] Appl. Environ. Microbiol.54: 397-404; Ingram, L. O, T. Conway, D. P. Clark, G. W. Sewell, and J.F. Preston [1987] Appl. Environ. Microbial. 53: 2420-2425.

Molecular genetics offers the potential to combine in a single organismthe pathway for anaerobic metabolism in pentose-utilizing entericbacteria, such as E. coli, the efficient pathway for ethanol productionfrom an ethanol producer such as Z. mobilis. Thus, expression of the Z.mobilis pdc gene in enteric bacteria such as E. coli, Erwiniachrysanthemi and Klebsiella planticola partially diverts the flow ofpyruvate to ethanol as a fermentation product by using low levels ofnative ADH activity. More efficient ethanol production and higherconcentrations of ethanol have been obtained with recombinant E. coliharboring the Z. mobilis genes encoding PDC and ADHII on a multi-copyplasmid. See Ingram et al. [1987], supra; Neale, A. D., R. K. Scopes,and J. M. Kelly [1988] Appl. Microbiol. Biotechnol. 29: 162-167. E. coliB (pLOI297) and E. coli ATCC 15224 (pLOI297) strains are superiorconstructs in terms of ethanol production and environmental hardiness.See Alterthum, F., and L. O. Ingram [1989] Appl. Environ. Microbiol. 55:1943-1948. These recombinant E. coli efficiently ferment glucose,lactose, and xylose to ethanol.

The recombinant E. coli described above achieved useful levels ofethanol production using plasmid-borne ethanol-production genes from Z.mobilis. Further, initial testing of ethanol production in prototypestrains was facilitated by placing the exogenous genes on a multi-copyplasmid. However, the exogenous genes were not completely stable becauseof the inherent instability of plasmids in the absence of selectivepressure to ensure their retention in the host cell. Due to plasmidincompatibilities, moreover, the use of a typical E. coli expressionplasmid for the ethanol-production genes precludes the most convenientmeans for introduction into a basic commercial ethanol-producer strainof additional exogenous genes for production of other selected products,such as valuable proteins.

SUMMARY OF THE INVENTION

The present invention pertains to recombinant host cells that expresschromosomally-integrated heterologous genes encoding useful polypeptidesat high levels. According to one aspect of the present invention, suchrecombinant cells are obtained by first inserting a heterologous DNAsegment encoding the desired polypeptide(s) into a host cell chromosomeunder the control of an endogenous promoter which is preferably a strongpromoter. Optionally, the transformed cells are then treated with amutagen. Finally, transformants are tested for expression ofheterologous genes, either by genetic selection or screening, to findthose having a mutation that causes increased expression of the insertedDNA segment resulting in an increase in production of each polypeptideencoded by the inserted DNA segment.

The chromosomally-integrated, heterologous genes and the mutation thateffects increased expression are extremely stable even in the absence ofconditions that select for retention of increased gene expression. Also,the engineered hosts are environmentally safer than conventional,plasmid-based recombinant production systems because they do not carrymobile genetic elements.

Specifically exemplified is a recombinant enteric bacterium, Escherichiacoli, that is capable of efficiently converting the entire range ofbiomass-derived sugars to ethanol using exogenous ethanol-productiongenes from an efficient ethanol producer, such as Zymomonas mobilis.These ethanol genes are stably integrated into the chromosome of therecombinant host under the control of an endogenous promoter for an E.coli pyruvate formate-lyase (pfl) gene. The chromosome of this hostfurther comprises a mutation that increases production of theethanol-production proteins according to this invention. Theserecombinant hosts can accommodate the usual E. coli expression plasmidsfor efficient co-production with ethanol of other desirable products,such as commercially valuable proteins. Accordingly, the residualbiomass from high volume fermentation of the bacteria of this inventionto produce ethanol may optionally provide one or more additionalhigh-value products in great abundance.

A further example is provided by recombinant Klebsiella oxytoca whichalso carries ethanol-producing genes from Z. mobilis stably integratedinto the host chromosome. This bacterium is capable of efficientlyproducing ethanol from cellobiose and cellotriose, as well as frommonomeric sugars.

More particularly, one aspect of the present invention relates to arecombinant host cell comprising a chromosome comprised of (a) aheterologous DNA segment under transcriptional control of a promoterendogenous to that host cell, where the DNA segment encodes a desiredpolypeptide; and (b) a mutation that causes increased expression of theheterologous DNA segment resulting in an increased production by therecombinant host cell of the desired polypeptide, compared to productionof that polypeptide by the recombinant cell in the absence of themutation. This increased expression of the heterologous DNA segment isretained in the absence of conditions that select for cells having suchincreased expression. In one preferred embodiment the chromosome of therecombinant host cell comprises a heterologous DNA segment that encodesa plurality of genes. Advantageously, among the plurality of genes is aselectable marker gene.

The present invention also relates to a cell strain that is the productof a process comprising the steps of

-   -   (a) providing a culture comprised of host cells comprising a        chromosome that encodes a promoter endogenous to these host        cells and a host gene for integration of heterologous genes that        is under transcriptional control of the endogenous promoter;    -   (b) transforming host cells in this culture with a heterologous        DNA segment comprising        -   (i) a plurality of genes including a selectable marker gene            and a gene encoding a desired polypeptide, and        -   (ii) sequences that are sufficiently homologous to the host            gene and properly located in the heterologous DNA segment to            enable integration into the host gene of the plurality of            genes encoded by the heterologous DNA segment by means of            homologous recombination;    -   (c) selecting for host cells in the above culture, or progeny        thereof, that express the selectable marker gene at a first        level;    -   (d) screening host cells selected in step (c), or progeny        thereof, to obtain host cells that produce the desired        polypeptide at an initial level;    -   (e) optionally exposing the host cells identified in step (d),        or progeny thereof, to a mutagen under conditions such that        mutations are created in the host cell chromosome; and then    -   (f) testing host cells produced in step (d) or step (e), or        progeny thereof, for host cells that produce the desired protein        at a level higher than the initial level, to obtain bacteria        having a mutation that causes increased expression of the        heterologous DNA segment.        This increased expression results in an increase in production        by the host cells of the desired polypeptide compared to        production of desired polypeptide by these host cells in the        absence of the mutation. Furthermore, enhanced expression of the        integrated heterologous DNA is retained in the absence of        conditions that select for cells having such increased        expression.

In preferred embodiments of the present invention, the above-describedprocess is further qualified as follows:

(i) In step (b), the heterologous DNA segment further comprises aplasmid which comprises a replicon that is temperature-sensitive forreplication.

(ii) In step (b), the process of transforming host cells also comprisesintroducing the heterologous DNA segment into host cells and growingthese host cells under conditions that select for cells that express theselectable marker gene at the first level. The cells are grown at atemperature that does not permit replication of the plasmid, resultingin integration of the heterologous DNA segment into the host gene of thechromosome by a homologous recombination event.

(iii) In step (c), the process of selecting for host cells furthercomprises growing the host cells that express the selectable marker geneat the first level under conditions that do not select for cells thatexpress the selectable marker gene. In particular, these host cells aregrown under these nonselective conditions at a first temperature thatdoes permit replication of the plasmid, resulting in spontaneousexcision from the host gene of the plasmid and the selectable markergene by a second homologous recombination event. The host cells are thengrown under the nonselective conditions at a second temperature thatdoes not permit replication of the plasmid, resulting in host cells thatretain the gene encoding the desired polypeptide in the absence of theselectable marker gene and the plasmid.

The basis of using nonselective conditions and a nonpermissivetemperature for plasmid replication to obtain according to this aspectof the invention is that integration of the plasmid replicon into thechromosome provides a slight inhibition of growth rate. Therefore,strains which have excised plasmid grow slightly faster than thoseretaining integrated plasmid, thus providing the basis for enrichment ofclones which have deleted the plasmid replicon and, coincidentally, theselectable marker gene.

In another preferred embodiment relating to this aspect of theinvention, the above-described process is further qualified such that(i) in step (b) the heterologous DNA segment comprises a closed circularDNA lacking an ability to replicate and (ii) in step (f) the process oftesting host cells comprises selecting for host cells produced in step(d) or step (e), or progeny thereof, that express the selectable markergene at a second level that is higher than the first level, and thenscreening these host cells that express the selectable marker gene atthe second level for host cells that produce the desired protein at alevel higher than the initial level. When the selectable marker geneconfers resistance to chloramphenicol, for example, host cells producedin step (d) or (e) of the above process may express the chloramphenicolresistance gene at a first level that confers resistance to at leastabout 20 μg/ml of chloramphenicol. In those embodiments where theprocess involves further selection for cells carrying mutations thatcause a second, higher level of expression of the chloramphenicolresistance gene, the second level of expression may confer resistance toat least about 100 μg/ml of chloramphenicol. Advantageously, selectionfor mutant cells with a higher level of expression of chloramphenicolresistance may be carried out using about 600 μg/ml of chloramphenicol.

A recombinant host cell of this invention can be a microbial cell, suchas an enteric bacterium. Illustrative of suitable enteric bacteria inthis regard are strains of Erwinia chrysanthemi, Escherichia coli,Klebsiella pneumoniae, and Klebsiella oxytoca.

The heterologous DNA segment which is chromosomally integrated into thegenome of the recombinant host, in accordance with the presentinvention, is preferably under the control of a strong endogenouspromoter, for example, the pyruvate formate-lyase (pfl) promoter.

In certain embodiments, the heterologous DNA segment encodes an alcoholdehydrogenase and a pyruvate decarboxylase from an organism thatproduces high levels of ethanol. Such enzymes are exemplified by analcohol dehydrogenase and a pyruvate decarboxylase encoded by genes fromZymomonas mobilis. In a preferred embodiment of the present invention,the recombinant host cell is able to produce ethanol by fermentation,for example, of glucose or xylose, with theoretical yields correspondingto conversion of at least about 90% or 100%, respectively, of addedsugar to ethanol. In some cases the observed yields of ethanol appear toexceed those possible based on the amount of added sugar, a resultreflecting co-catabolism of complex nutrients to pyruvate and thus toethanol.

In accordance with an additional aspect of the present invention, arecombinant host cell as described above is provided that contains achromosome comprising a further mutation, particularly a mutation in afumarate reductase (frd) gene, that impairs succinate production,reducing production of acid which can inhibit ethanol production.

A recombinant host cell within the present invention optionally maycomprise a further mutation that impairs recombination in the cell, tomake the cell safer environmentally by reducing its ability to interactwith mobile genetic elements. In one embodiment, the mutation thatimpairs recombination comprises a mutation in a recA gene.

A recombinant host cell of this invention can be produced by variousprocesses in addition to those particularly exemplified here. Exemplaryof such processes are genetic-engineering methods for insertion of aheterologous DNA segment into a predetermined location on a host cellchromosome, and for generation and identification of mutations whichenhance expression. In particular, standard molecular genetic analysesof a recombinant microbial strain of the present invention will enableone to employ other methods to make recombinant host cells whichlikewise fall within the present invention. For example, knowledge ofthe DNA-sequence changes associated with a particular mutation effectedin accordance with the present invention enables the design, synthesisand insertion into a host chromosome of a heterologous DNA segmentcontaining not only genes coding for desired proteins but also apromoter and a mutation effecting high-level expression of the genes.Such a genetic construct, comprising a heterologous DNA segment thatincludes an appropriate mutation and promoter with the desired genes, istherefore another embodiment within the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram illustrating construction of anintegration vector (pLOI543), containing a temperature-conditionalreplicon (pSC101; Hamilton, C. M., M. Aldea, B. K. Washburn, P.Babitzke, and S. R. Kushner [1989] J. Bacteriol. 171: 4617-22) for theintegration of ethanol-production genes (Z. mobilis pdc and adhB genes)and a chloramphenicol resistance gene into a pfl gene of an E. colichromosome. Abbreviation: Klenow, convert to blunt by filling bases inoverhang region using the Klenow fragment of E. coli DNA polymerase I.

FIG. 2 is a schematic diagram illustrating construction of a plasmid(pLOI510) for the integration of Z. mobilis pdc and adhB genes into apfl gene of an E. coli chromosome using a vectorless, circularized DNAfragment. Abbreviations: Klenow, convert to blunt by filling bases inthe overhang region using the Klenow fragment of DNA polymerase I; Cir.Frag., circularized SalI fragment from pLOI510 which has been elutedfrom an agarose gel and ligated to form closed circles.

FIG. 3 illustrates ethanol (A, C, E, G) production and growth (B, D, F)during batch fermentation. A and B. Fermentation of 10% glucose.Symbols: ●, plasmid-based ethanol production strain ATCC11303 (pLOI297);◯, chromosomally-integrated ethanol production strain lacking anexpression enhancing mutation, KO2. C. and D. Fermentation of 10%glucose. Symbols: ◯, a chromosomally-integrated strain lacking aspontaneous expression enhancing mutation, KO3; ●,chromosomally-integrated strain containing a spontaneous expressionenhancing mutation, KO4; ▴, KO4 supplemented with 22 mM sodium acetate.E. and F. Fermentation of 8% xylose. Symbols: ●, KO4; ▴, KO11, a furthermutant of KO4, lacking fumarate reductase activity (frd); □, KO12, afurther mutant of KO11, carrying a recA mutation. G. Fermentation byKO20, a chromosomally-integrated strain with an induced mutation.Symbols: ▪, 10% glucose, ●, 8% xylose.

FIG. 4 illustrates fermentation of 10% glucose by KO11 (frd) without pHcontrol. A. Growth (●) and ethanol production (◯). B. pH of broth (Δ).

FIGS. 5 and 6 illustrate the ethanol production by recombinant strainsof K. oxytoca M5A1. FIG. 5 illustrates the ethanol production fromglucose (100 g/liter) by: (•) strain M5A1(pLOI555), (▴) strain P2containing integrated PET genes, and (▪) strain B1 containing integratedPET genes. FIG. 6 illustrates the ethanol production from cellobiose(100 g/liter) fermentation by strain P2. (▴), ethanol; (Δ), cell mass.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention relates to a recombinant host cell comprising achromosome comprised of (a) a heterologous DNA segment undertranscriptional control of a promoter endogenous to that microbial cell,where the DNA segment encodes a desired polypeptide; and (b) a mutationthat causes increased expression of the heterologous DNA segmentresulting in an increase in production by the host cell of the desiredpolypeptide compared to production of that polypeptide by the host cellin the absence of this mutation. Enhanced expression of the heterologousDNA segment is retained in the absence of conditions that select forcells having such increased expression.

Definitions: In the present context, “heterologous” DNA segment meansthat the DNA segment contains a sequence that is different from thesequence in the corresponding position downstream from the endogenouspromoter in the chromosome of the cell into which the heterologoussegment has been inserted. Thus, the heterologous DNA segment of thepresent invention includes a DNA segment taken from one location in ahost chromosome and inserted into another location (under control of anendogenous promoter other than the one naturally associated with thatsegment) as well as a DNA segment from another organism. The nucleotidesequence of the heterologous DNA segment encodes one or more structuralgenes and is derived from any genetic source, including, for example,eukaryotic or prokaryotic cellular or viral genomes, or from artificialcoding sequences created by genetic engineering.

The heterologous segment is under the transcriptional control of anendogenous promoter by virtue of being integrated into a host cellchromosome on the downstream (3′) side of the promoter. As previouslynoted, the endogenous promoter is preferably a “strong” promoter, in thesense that it provides a high level of gene expression in relation to amore typical microbial promoter, such as the lactose operon (lac)promoter of E. coli. Among strong promoters are those that include asingle site for promoter activity, i.e., for binding RNA polymerase anddirecting the enzyme to the correct transcriptional start site, andthose that include a plurality of such sites. Illustrative of the former(single-site) category of strong promoters is the well-known tryptophan(trp) promoter. The latter (multi-site) category is exemplified by thepromoter for a pyruvate formate-lyase (pal) gene, homologous variants ofwhich are found in E. coli and other enteric bacteria. Pyruvateformate-lyase is normally expressed at a high level, particularly underthe anaerobic conditions which pertain during fermentation withoutforced aeration. The pfl promoter in fact includes seven sites forpromoter activity, as reflected, for example, in sequence analyses ofthe pfl mRNA and in “footprinting” data indicating where RNA polymeraseis bound, or in “primer extension” analyses indicating the 5′ end of amRNA.

In this description, “mutation” denotes a relatively permanent change inhereditary material, typically involving a biochemical change in thecodons that make up genes but also possibly involving a physical changein chromosome relations. A mutation suitably employed according to thepresent invention causes increased expression of the heterologous DNAsegment, resulting in enhanced production by the host cell of eachpolypeptide encoded by that segment, relative to production of eachpolypeptide by the host cell in the absence of the mutation. Theincreased expression thus achieved is retained in the absence ofconditions that select for cells having such increased expression. Forexample, in bacterial strains having mutations, according to the presentinvention, that increase expression of both antibiotic resistance andethanol-production enzymes integrated into a chromosome, no loss ofhigh-level expression of the integrated genes is detectable after asmany as 68 population doublings, even without selection with antibiotic.

Transformed host cells bearing a mutation according to the presentinvention may be detected conveniently by means of the increasedexpression of one or more polypeptides encoded by the inserted DNAsegment, according to standard genetic methodology. Increased expressionof a polypeptide may be determined by direct detection of a polypeptideor inferred from an activity attributable to that polypeptide.Advantageously, one of the genes encoded by the inserted DNA segment canbe a selectable marker gene, for example, a gene which confersantibiotic resistance to the host. In this case, mutant cells withincreased expression of the antibiotic resistance gene may be selectedconveniently using antibiotic concentrations above a first level thatinhibits host cells lacking a mutation that increases expression of theheterologous DNA segment.

More particularly, it has been discovered that mutations suitable foruse in the present invention can be obtained reliably as spontaneousmutants or by induction via conventional mutagenesis, followed byselection or by screening for cells having the desired phenotype ofhigh-level expression of integrated genes. Thus, suitable mutations incertain bacteria are found to arise spontaneously at a frequency on theorder of 10⁻⁴ to 10⁻⁵ upon selection for cells having increasedexpression of an integrated chloramphenicol resistance gene, such thatcell growth occurs at a concentration of antibiotic 30 times higher thanthat tolerated in the absence of such a mutation. Similarly, aftertreatment of certain bacterial cells with a mutagen, suitable mutationscan be obtained at a frequency of about 0.5-1×10⁻⁴ of the survivors ofmutagenesis by screening for cells having expression of ethanol-relatedenzymes at levels about ten times higher than in the absence of suchmutagenic treatments.

Mutations suitable for use in the present invention actually causeoverexpression of inserted genes to the extent that polypeptideproduction from a single copy of a gene chromosomally inserted,according to this invention, is comparable to production achieved withmultiple copies of the same gene carried on a multi-copy plasmid. Forexample, bacterial cells carrying such a suitable mutation can expressintegrated genes, say, for chloramphenicol resistance and ethanolproduction, at levels that are functionally equivalent to a cellcontaining 30 to 300 copies of the same genes on a multi-copy plasmid.

A molecular basis has not been determined for mutations identified, asdescribed herein, for use according to the present invention.Nevertheless, the magnitude of the increase in gene expression (on theorder of about ten fold, for example) that is obtained in a singlescreening or selection step is substantially greater than that expectedfor a mutation mechanism involving gene duplication. To achieve acomparable increase in selectable gene expression by means of geneduplication typically requires repeated selection steps withprogressively higher levels of selective agent. Moreover, the low (infact, undetectable) frequency of reversion of the present mutations alsomilitates against gene duplication as a basis for these mutations. Infact, it is known that mutations which produce increased gene expressionby means of tandem gene repetition are typically unstable and cannot beretained in the absence of conditions that select for cells having suchincreased gene expression, in contrast to mutations of the presentinvention. Conversely, the observed frequency of suitable mutationsarising spontaneously, according to the present invention, is compatiblewith a causal mechanism involving point mutations.

Selection of mutations that enhance expression of integrated genes usinga selectable marker gene may be performed pursuant to the presentinvention in any type of host cell for which practical genetic methodsare known in the art for selecting clones having mutations that areinducible at a frequency on the order of at least about 10⁻⁴ persurvivor of mutagenic treatment. Similarly, screening for mutant hostcells pursuant to the present invention requires practical methods forpreparation of sufficient numbers of clones, each comprising enoughcells for detection of high-level expression of a desired integratedgene by a method appropriate to the particular desired gene.

Such selection or screening for suitable mutations is most easilyeffected with host cells which are readily culturable in largequantities, as in the case of prokaryotic (bacterial) and yeast cells.For purposes of this description, such a host is denoted a “microbialcell,” and the strain it comprised is a “microbial strain.” Under thisrubric of microbial cells, enteric bacteria like Erwinia chrysanthemi,Escherichia coli and Klebsiella planticola are particularly attractivehosts because they are capable of utilizing a wide variety of sugars,including pentoses and lactose. While microbial cells are preferredhosts, the present invention also contemplates the use of other types ofcellular hosts, including fungal cells and eukaryotic (animal, insectand plant) cells, into which a heterologous DNA segment can be insertedunder control of an endogenous promoter. Thus, the host cell need onlybe amenable to a selection or screening regimen for cells havingincreased expression of the integrated heterologous DNA, as describedabove, thereby to obtain suitable enhanced-expression mutants.

Embodiment relating to ethanologenic E. coli: An illustrativeapplication of the present invention involves the integration of Z.mobilis genes for ethanol production into the pfl region of an E. colichromosome. The pfl gene is central to normal fermentative metabolism,catalyzing the conversion of pyruvate to formate plus acetyl-CoA andproviding an essential source of acetyl units for biosynthesis.Insertional inactivation of this gene thus represents inhibition of acompeting branch point for the diversion of pyruvate away fromproduction of ethanol by the microorganism. Additional geneticimprovements are also described which eliminate succinate production andinactivate the recA gene.

These recombinant bacteria are useful for the manufacture of recombinantpolypeptides as co-products during ethanologenic fermentation of biomasssugars. As a result of integration into the chromosome, the stability ofthe ethanol production trait is improved significantly compared tocomparable plasmid-based systems for the purposes of industrial-scalefermentation to produce ethanol: 100% retention of the ethanol genesafter 68 generations compared to only 97% with a comparableplasmid-based construct. Also, elimination of the plasmid carryingethanol-production genes allows insertion of plasmids for producingco-products without interference with the ethanol production ability.

Biological deposits: The following cultures have been deposited with theAmerican Type Culture Collection (ATCC), 12301 Parklawn Drive,Rockville, Md., 20852, USA. Table 1 lists the accession numbers assignedto the cultures by the repository. TABLE 1 Biological deposits CultureAccession Number E. coli (pLOI510) ATCC 68484 E. coli (pLOI543) ATCC68485 E. coli KO4 ATCC 55123 E. coli KO11 ATCC 55124 E. coli KO12 ATCC55125 E. coli KO20 ATCC 55126

The subject cultures have been deposited under conditions that assurethat access to the cultures will be available during the pendency ofthis patent application to one determined by the Commissioner of Patentsand Trademarks to be entitled thereto under 37 CFR §51.14 and 35 USC§122. The deposits are available as required by foreign patent laws incountries wherein counterparts of the subject application, or itsprogeny, are filed. It should be understood, however, that theavailability of the deposits does not constitute a license to practicethe subject invention in derogation of patent rights granted bygovernmental action.

Further, the subject culture deposits will be stored and made availableto the public in accord with the provisions of the Budapest Treaty forthe Deposit of Microorganisms, i.e., they will be stored with all thecare necessary to keep them viable and uncontaminated for a period of atleast five years after the most recent request for the furnishing of asample of the deposit(s), and in any case, for a period of at least 30(thirty) years after the date of deposit or for the enforceable life ofany patent which may issue disclosing the cultures. The depositoracknowledges the duty to replace the deposits, should the depository beunable to furnish a sample when requested, due to the condition of thedeposit. All restrictions on the availability to the public of thesubject culture deposits will be irrevocably removed upon the grantingof a patent disclosing them.

Integration of Z. mobilis pdc and adhB genes into the E. coli Bchromosome: Two approaches embodied in examples set out below representdifferent strategies which can be employed, in accordance with thepresent invention, to construct a bacterial strain, such as E. coli(ATCC 11303), in which a foreign gene (here, the Z. mobilis genes forethanol) is integrated into a chromosomal gene, illustrated by the pflgene. The first approach entails the use of a derivative of thetemperature-conditional integration vector developed by Hamilton et al.See Hamilton, C. M., M. Aldea, B. K. Washburn, P. Babitzke, and S. R.Kushner [1989] J. Bacteriol. 171: 4617-22; and Example 3, infra.

Thus, as shown in the examples, ATCC 11303 was transformed with atemperature-dependent replication plasmid, pLOI543, followed bytemperature-based selection and enrichment for two homologousrecombination events, 64.5% of the colonies were sensitive tochloramphenicol (Cm), indicating loss of plasmid of these Cm-sensitiveclones, 5.9% formed pink colonies on aldehyde test plates indicating thepresence of Z. mobilis ADHII. Two of these clones having retainedintegrated ethanol-production genes but having lost the Cm-resistancegene and plasmid were selected for further study and designated KO1 andKO2.

Via a second approach, reflected in Example 4, a Cm-resistanttransformant was obtained using the circularized SalI fragment fromplasmid pLOI510 and was designated KO3. In this approach no replicatingplasmid is introduced into the cell; only the ethanol-production genesand associated Cm-resistance gene are integrated into the chromosome.The KO3 clone also formed pink colonies identical to KO1 and KO2 onaldehyde indicator plates. Control ATCC 11303 formed white colonies(negative), while ATCC 11303 (pLOI297) carrying Z. mobilis ethanol geneson a plasmid formed intensely red colonies indicating a high level of Z.mobilis gene expression.

Subsequently, isolation of vector from these three recombinants wasattempted by the alkaline SDS lysis method. No vector was visible inagarose gels of these preparations which had been stained with ethidiumbromide. These preparations were also tested in transformationexperiments with TC4 as the host with selection for Cm resistance. Notransformants were recovered further confirming the absence of vector.

Fermentation by strains KO1, KO2, and KO3: FIG. 3 A and B shows acomparison of fermentations by strains KO2 and ATCC 11303 (pLOI297) with10% glucose. Fermentation by strain KO1 was identical to that by KO2 andis not presented. These new constructs were very inefficient producersof ethanol as indicated by the low volumetric productivity and ethanolyield (Table 2). Growth was limited to less than half that of ATCC 11303(pLOI297) and only 4 g/liter of ethanol was produced after 72 hours.Although strain KO3 was slightly better than KO1 and KO2, it remained avery poor producer of ethanol (FIG. 3 C-D; Table 2). TABLE 2 Ethanolproduction from glucose and xylose by recombinant strains of E. coli(ATCC11303). 10% Glucose Parameter^(a) pLOI297b KO1 KO2 KO3 KO4 KO5 Base1.1 5.7 6.3 5.5 1.3 1.4 (mmoles/g sugar) Ethanol 48.8 4.0 4.0 10.4 52.852.8 Yield (g/liter) Ethanol 0.52 0.05 0.05 0.13 0.56 0.56 Yield (g/gsugar) Theoretical 101 10 10 26 110 110 Yield (%) Vol. Prod.^(c) 1.9 0.30.3 0.4 1.5 1.5 (g/liter*h) 30-h 41.8 3.2 3.2 6.4 36.0 36.0 Ethanol(g/liter) Cell Yield .048 .021 .021 .028 0.44 .040 (g/g sugar) 10%Glucose KO10 KO11 KO11 KO12 KO4 + Parameter^(a) (recA) (frd) (frd)^(d)(frd, recA) Ac° Base 1.1 0.6 0 1.1 1.0 (mmoles/g sugar) Ethanol 51.252.8 38.8 54.4 54.4 Yield (g/liter) Ethanol 0.54 0.54 0.39 0.57 0.57Yield (g/g sugar) Theoretical 107 107 76 112 112 Yield (%) Vol. Prod.1.8 1.7 1.4 1.2 1.6 (g/liter*h) 30-h 38.0 38.0 29.0 30.4 38.5 Ethanol(g/liter) Cell Yield .041 .042 .041 .035 .045 (g/g sugar) 8% Xylose KO11KO12 Parameter^(a) pLOI297^(b) KO4 (frd) (frd, recA) Base 1.4 1.6 0.50.64 (mmoles/g sugar) Ethanol 36.0 36.0 41.6 40.8 Yield (g/liter)Ethanol 0.47 0.47 0.53 0.53 Yield (g/g sugar) Theoretical 94 94 104 103Yield (%) Vol. Prod. 1.0 1.1 1.3 1.1 (g/liter*h) 30-h 30.0 25.6 30.426.0 Ethanol (g/liter) Cell Yield .050 .047 .051 .048 (g/g sugar)^(a)Calculations based on total sugar initially added.^(b)ATCC11303(pLOI297) has plasmid-borne ethanol genes.^(c)Vol. Prod., volumetric Productivity = yield/time^(d)Fermentation conducted without pH control.^(e)Supplemented with 3 g/liter sodium acetate (22 mM finalconcentration).

Large volumes of base were consumed for maintenance of pH duringfermentations by these three new strains, indicating excessiveproduction of acidic fermentation products.

Enhancement of expression of integrated pdc and adhB genes by mutations:The pink phenotype observed on aldehyde indicator plates appeared toindicate insufficient expression of Z. mobilis genes for ethanolproduction. A mutation that causes increased expression of theintegrated genes in KO2 was obtained by screening for dark red phenotypeon aldehyde indicator plates after mutagenesis with ethyl methanesulfonate under standard conditions well known in the art. Approximately200 plates were analyzed with 200 to 400 colonies per plate. Four darkred clones were isolated, the increased color being indicative ofincreased expression of ADHII. One of these was designated strain KO20.

Selection for resistance to high levels of Cm was used to enrich forspontaneous mutants of KO3 to determine whether mutants expressingantibiotic resistance at a higher level also expressed the otherinserted genes at a higher level. Serial dilutions of an overnightculture were plated on Luria agar plates containing 2% glucose and 600μg/ml of Cm. Large raised colonies which are indicative of high-levelexpression of Z. mobilis pdc and adhBita genes were observed afterovernight incubation at a frequency of approximately 1 per 100,000plated cells. All of these colonies exhibited a dark red phenotype onaldehyde indicator plates identical to ATCC 11303 (pLOI297), theplasmid-based construct which is an excellent ethanol producing strain.Two mutants were retained for further study, strains KO4 and KO5. Thelack of vector in these strains was again confirmed by the failure ofDNA preparations from KO4 and KO5 to transform TC4 during selection forCm resistance and by the absence of vector DNA in agarose gels stainedwith ethidium bromide.

Fermentation by strains KO4, KO5 and KO20: FIG. 3 C and D illustrate thefermentation of 10% glucose by KO4 and KO5, two strains in whichspontaneous mutations that enhance expression of integratedethanol-production genes were co-selected by selection for resistance tohigh levels of an antibiotic. Both strains were identical and onlyfermentation by KO4 has been plotted. Growth, cell yield, and ethanolyield by these improved constructs were almost equivalent to those ofthe plasmid-based construct, ATCC 11303 (pLOI297). See Table 2. Althoughthe rate of ethanol production as indicated by volumetric productivitiesfrom the early stages and by the level of ethanol achieved after 30hours was somewhat slower than ATCC 11303 (pLOI297), theoretical yieldswith KO4 and KO5 were higher and exceeded 100% based on added glucose.This higher yield during slower fermentation reflects co-catabolism ofcomplex nutrients to pyruvate and thus to ethanol. These complexnutrients serve as the primary nitrogen source for biosynthesis.Continued denitrification caused an increase in the pH with KO4, KO5 andATCC 11303 (pLOI297) after sugars were exhausted. The resulting rise inpH provides a convenient method to monitor sugar exhaustion.

The fermentation of 8% xylose by strain KO4 and ATCC 11303 (pLOI297),were also compared (FIG. 3 E and F). KO4 was equivalent to theplasmid-based strain in ethanol yield, although the rate of ethanolproduction was slightly slower as evidenced by the ethanol level after30 hours (Table 2).

FIG. 3 G illustrates the fermentation of 10% glucose and 8% xylose bystrain KO20 which comprises an induced mutation that enhances expressionof integrated ethanol-production genes. Fermentation of both sugars bythis strain carrying an induced mutation was essentially the same as forstrains KO4 and KO5 which carry spontaneous mutations selected by highlevels of antibiotics.

Effect of added acetate: During anaerobic growth, the pfl gene producthas been shown to be the primary route for acetate production and theprimary source of acetyl-CoA for biosynthesis (7,17). Insertionalinactivation by the Z. mobilis ethanol-pathway genes may lead to anacetate deficiency for lipid synthesis in derivatives of ATCC 11303.Supplementation with sodium acetate was found to improve the rate ofethanol production with a small increase in yield (FIG. 3 C and D; Table2).

Expression of Z. mobilis enzymes in recombinant E. coli: The specificactivity of Z. mobilis PDC was measured in French press extracts ofselected recombinant strains. PDC is a relatively thermostable enzymeand activities were measured in extracts after heat inactivation ofnative E. coli activities which complicate such measurements. Noactivity was detected in the control, strain ATCC 11303. Both KO2 andKO3 produced low levels of activity, 0.2 U/mg protein. Ten-fold higheractivity was present in extracts of KO4 (2.1 U/mg protein), the highCm-resistant mutant. The level of PDC in KO4 was almost equivalent tothe level produced in the plasmid-based construct, ATCC 11303 (pLOI297),and similar to that found in native Z. mobilis (Ingram, L. O., and T.Conway [1988] Appl. Environ. Microbiol. 54: 397-404).

Protein extracts from these strains were also examined by SDS-PAGE.Numerous changes in proteins were observed between ATCC 11303 andrecombinant derivatives in addition those attributable to Z. mobilisgenes. KO4 and ATCC 11303 (pLOI297) contained higher levels of a proteinband in the 60,000 MW region corresponding to the size of PDC than werepresent in KO2 and KO3. This band was absent in the ATCC 11303 control.Many endogenous proteins were found in the 38,000 MW region where Z.mobilis ADHII would be found, obscuring differences in expression.

Organic acid production by E. Coli ATCC11303 constructs: Considerableamounts of base were consumed by ethanologenic strains of E. coli duringfermentations (Table 2) indicating the production of acids asco-products. As shown in Table 3, high levels of three acids areproduced by the ATCC 11303 parent and by KO3. Although ATCC 11303(pLOI297) and KO4 produced lower levels of acetic and lactic acids,succinate production by KO4 remained high. To eliminate succinateproduction, a frd mutation was introduced by insertion of a Tn10 vectorusing standard genetic methods, including selection for a tetracyclineresistance marker of Tn10, followed by elimination of Tn10 using fusaricacid selection, resulting in an frd deletion due to imprecise excisionof Tn10. The resulting strain (KO11) lacked fumarate reductase activity.

This frd mutation reduced the level of succinate produced during glucosefermentation to 3% that of the parent, KO4 (Table 3). However, withouttitration of acid during fermentation, this low level of acid productionwas still sufficient to reduce the pH of fermentations run withouttitration and, therefore, to reduce ethanol productivity from glucose(FIG. 4, Table 2).

However, with acid titration, the elimination of fumarate reductase didimprove the rate of ethanol production, cell yield, and ethanol yieldfrom xylose (Table 2). With titration, volumetric productivity fromglucose was also increased by this mutation, although ethanol yieldremained essentially the same (Table 2). TABLE 3 Production of acidicfermentation products^(a). Organic Acid (mM) Acetic Lactic SuccinicRecombinant^(b) acid acid acid ATCC11303 (None) 66 641 58 ATCC11303(pLOI297) 21 32 49 (plasmid-borne) KO1 (integrated, 18 673 62 nomutation) KO2 (integrated, 18 602 57 no mutation) KO3 (integrated, 69525 66 spontaneous) KO4 (integrated, 22 29 70 spontaneous) KO10 (KO4,recA) 18 20 73 KO11 (KO4, frd) 14 32 2 KO12 (KO4, recA, frd) 6 40 2 KO20(integrated, 4 60 2 induced)^(a)Average of two fermentations with 10% glucose, sampled after 72 h.^(b)Notes in parentheses indicate status of exogenous ethanol genes andrelated mutations that enhance ethanol production.

Comparison of the stability of chromosomally-integrated andplasmid-based genes in E. coli. ATCC 11303 (pLOI297), KO4 and KO5 weregrown in the Luria broth containing 10% (w/v) glucose without antibioticselection for up to 68 generations at 30o C. Cultures were plated onselective and nonselective plates and on aldehyde indicator plates after48 and 120 hours to determine the ratio of Cm-resistant CFU to total CFUas well as the proportion of ethanol-producing colonies. Table 4 showsthat the inserted genes of KO4 and KO5 were stably maintained as achromosomal insertion. TABLE 4 Stability of chromosomally-integratedgenes compared to plasmid-based genes in E. coli B^(a) Percentageretaining traits production Recombinant (number of generations) KO4 100(38.5) 100 (68.5) KO5 100 (38.7) 100 (68.7) Tc E. coli B (PLOI297) 100(37.3)  97 (67.3)^(a)Transformed E. coli B were grown at 30° C. in Luria broth containing10% (w/v) glucose without antibiotic selection. Tc resistance andethanol genes were coordinately lost from the plasmid-based construct.

Strains KO4 and KO5 in which the genes for ethanol production have beenintegrated into the chromosome are superior to the prior construct withplasmid pLOI297, see Alterthun, F., and L. O. Ingram [1989] Appl.Environ. Microbiol. 55: 1943-1948, in terms of retention of therecombinant trait, ethanol production. Even when the rate of plasmidloss is low, as in E. coli strains carrying pLOI297, strains carryingintegrated exogenous genes according to the present invention offerconsiderable advantages over comparable plasmid-based strains for thepurpose of commercial ethanol production which involves the scaling upof small cultures to millions of gallons of fermentation broth.

Effect of recA mutation: A recA mutation was also introduced into KO4and KO11 so that these strains could be used as hosts for recombinantplasmids. The resulting strains were designated KO10 and KO12,respectively. The recA mutation did not affect the production of acetic,lactic, or succinic acids but did reduce growth and slow fermentation(Table 2) in the recA, frd mutant (KO12). The basis of this effect isunknown, and it may represent secondary mutations which have beencreated during construction.

Embodiment relating to ethanologenic Klebsiella oxytoca: A furtherillustrative application of the present invention is provided by theintegration of Z. mobilis genes for ethanol production into thechromosome of K. oxytoca. This can be carried out, for example, by arecombination protocol analogous to that described above for E. coli. K.oxytoca and E. coli are closely related, and therefore DNA fragmentscarrying the Z. mobilis pyruvate decarboxylase and alcohol dehydrogenase(PET) genes within the E. coli pfl gene can be used as a source of DNAto promote the desired recombination event. Briefly, E. coli plasmid orchromosomal DNA carrying both the PET genes and a selectable marker(such as cat) within the pfl gene are digested with restriction enzymeswhich cut within the pfl gene only. The resulting fragments arecircularized and used as transforming DNA for integration experiments asdescribed above for E. coli. Screening of transformants for the presenceof the selectable marker and the absence of transforming plasmid allowsselection of bacteria in which the transforming DNA has becomeintegrated into the host plasmid. The preparation of recombinant K.oxytoca containing stably integrated PET genes is further illustrated inExample 9 below.

Genes that can be chromosomally inserted according to the presentinvention: The present invention can be practiced using genes other thanthe specific adh and pdc genes exemplified herein. It is now wellestablished that the enzymes of glycolysis exhibit a great deal ofconservation of primary sequences. See, for example, Conway, Sewell, andIngram [1987] J. Bacteriol. 169: 5653-5662. This high level ofconservation enables those skilled in the art to isolate functionallyequivalent, genetically related enzymes from other organisms usingprimary information from one or more members of an enzyme family.Indeed, just such an approach has been used successfully to clone thepyruvate decarboxylase gene from maize using the current inventors'information on the Z. mobilis pdc and the pdc of S. cerevisiae to designa DNA probe. See Kelly, P. M. [1989] Plant Molecular Biology 13:213-222. Alternative strategies using entire genes as probes can also beused. Thus, for purposes of this invention, it does not matter if thepyruvate decarboxylase activity is provided by a gene from Z. mobilis,as exemplified in the subject invention, or from genes which specify thesame enzymatic activity from corn (which has been cloned and sequenced),yeasts or another organism. Also, in order to practice the invention, itdoes not matter if the alcohol dehydrogenase activity is provided from agene from a horse, yeast, human or insect, or from another bacterialgene. Since expression of alcohol dehydrogenase activity can be observeddirectly on aldehyde indicator plates, sequence information would notnecessarily be the best approach to the isolation of additional genesencoding proteins which exhibit this enzymatic activity. However,whether or not sequences are to be used for such isolation is not reallycritical. Indeed, many alcohol dehydrogenase genes are already in handand well described in many papers.

Z. mobilis contains two genes encoding functional alcohol dehydrogenasegenes. The one which has been exemplified here, adhB, is evolutionarilyrelated to a butanol (alcohol) dehydrogenase from Clostridiumacetobutylicum, propanediol (alcohol) oxidoreductase from E. coli, andADHIV alcohol dehydrogenase from Saccharomyces. All have been clones andsequenced. The second Z. mobilis gene encoding alcohol dehydrogenase,adhA, is a zinc alcohol dehydrogenase and has recently been cloned andsequenced by us. This adhA is evolutionarily related to the typicalalcohol dehydrogenases described in animals, plants, and the dominantgene in yeasts based upon comparisons of primary structure deduced fromnucleotide sequences which are available for all. We have found thatthis adhA gene substitutes quite nicely for the original adhB gene, asexpected pursuant to the present invention.

The synthesis of a protein with pyruvate decarboxylase activity(pyruvate converted to acetaldehyde plus carbon dioxide) can be observeddirectly on aldehyde indicator plates. The expression of alcoholdehydrogenase activity can also be directly observed on aldehydeindicator plates. Therefore, sequence information would not necessarilybe the best approach to locating other adh or pdc genes, although, asdescribed above, sequence information from our work was used recently toisolate the corn pdc gene. Thus, many other pdc and adh genes whichprovide a functional equivalent can be isolated from other organisms. Itis entirely predictable that these other genes would be suitablereplacements for the Z. mobilis pdc and adh genes available for use, andother such genes can be identified either by use of the current genes asprobes or, more preferably, by observing activity on indicator plates.

More generally, many genes other than ethanol-production genes could beincorporated into a chromosome and expressed according to the presentinvention. These include essentially any desired polypeptide that can beexpressed in a recombinant host, for example, are genes encodinginsulin, growth hormones, and commercially important enzymes.

Utility of exemplary bacteria: Certain bacteria and other simpleorganisms are capable of actively metabolizing a wide variety ofsubstrates, including hexoses, pentoses, and lactose. Thischaracteristic makes E. coli an attractive host for recombinant DNAproduction methods. The invention described here permits the use ofrecombinant bacterial strains for the economical production of ethanolfrom a variety of biomass sources, particularly from under utilizedsources of biomass such as hemicellulose (comprising xylose, arabinose,and other sugars), which represents a major portion of wood and inedibleplant parts, and whey (lactose). Also, organisms with specialcapabilities, such as production of extracellular enzymes for thedegradation of complex polymers, can be converted to ethanol producersaccording to the present invention.

Thus, one aspect of the present invention provides an improved organismfor the production of ethanol. An E. coli cell has been transformed suchthat Z. mobilis genes coding for adh and pdc have been incorporated intothe host chromosome. In the past, ethanol-production capability has beenconferred on E. coli by transformation with a plasmid comprising the twogenes needed for ethanol production. The success of these previoustransformations resulted from the very high levels of theethanol-producing enzymes which were made from multiple copies of thegenes, typically, 30 to 300 copies per cell.

The initial constructs described here in which single copies of pdc andadh genes were integrated into the host chromosome provided insufficientlevels of PDC and ADH to divert metabolism to ethanol and duplicate thelevel of functionality provided by multiple copies of these genes on aplasmid. Subsequently, spontaneous or induced mutations described aboveproduced cells which simultaneously increased expression of allchromosomally-integrated genes, including that for chloramphenicolresistance, when present, and those for ADH and PDC. These mutantsproduce high levels of ethanol-production enzymes and are thusfunctionally equivalent to previous bacteria containing multi-copyplasmids. In the case of spontaneous mutations in a strain carrying aCm-resistance marker gene as well as ethanol genes, the result wherebyincreased expression of the Cm-resistance gene also indicated increasedexpression of the ethanol-related enzymes is particularly surprisingbecause the Cm-resistance gene can include its own promoter and can evenbe downstream from a transcription terminator of an adhB gene.

Another aspect of this invention concerns the use of the recombinantethanol-producing bacteria for the efficient production of recombinantproteins; that is, the recombinant cells can be further transformed withgenes coding for useful proteins. These additional genes can also beincorporated into a chromosome, or they can be plasmid-borne. Indeed,since high-level expression of the integrated (in this example,ethanol-production) genes, in accordance with the present invention,obviates the need for a plasmid to carry them, recombinant cells of thepresent invention are particularly well-suited to receive and maintainadditional genes carried on a plasmid. An additional mutation, such as arecA mutation, that inhibits homologous recombination will enhance theenvironmental safety of such hosts in the context of recombinant proteinproduction.

It should be noted that the accumulation of organic acids from sugarmetabolism is generally regarded as a consequence of fermentation duringanaerobic growth. But appreciable quantities of acetate are generallyproduced by E. coli even during rapid agitation under aerobicconditions. The production of acetate is progressive from the earlieststages of growth and is not limited to the later stages, when celldensity is high. This acid production from glucose even under aerobicconditions serves to limit growth in broth and on solid medium, asdemonstrated by the increased final cell density in medium supplementedwith phosphate buffer.

The conversion of a host organism to ethanolic fermentation can be usedto enhance the production of a variety of recombinant products. Themaintenance of function in these products is related to the pH of thebroth during growth in dense culture. The extent of this acidificationper unit of cell protein is minimized by the production of ethanolrather than of organic acids. Oxygen transfer is frequently a majorlimitation during the growth of dense cultures of microorganisms, and itis this limitation which results in acid production and pH drift of thegrowth medium. In recombinants producing ethanol as a fermentationproduct, the ethanologenic enzymes divert part of the pyruvate fromglycolysis to acetaldehyde and reoxidize NADH to produce ethanol, a lessdamaging product of metabolism. Strains containing both functionalrespiratory chains for oxidative phosphorylation and ethanol-productionenzymes can be grown to even higher cell densities because of theoperation of both systems during the regeneration of NAD⁺ a reduction inacidic waste products. Such inherent flexibility results in lessstringent process-control requirements, as well as increased yields ofrecombinant products.

The ethanol-producing bacterial strains of this invention are thussuperior hosts for production of recombinant proteins under anaerobicconditions with minimal acid production. Many recombinant proteinscontain cysteine or disulfide bridges, and proper folding or reactionsof these is an essential feature to form the active enzyme. Sinceformation of disulfide bonds is promoted by oxygen, synthesis of suchproteins under anaerobic conditions provides less opportunity forimproper folding prior to isolation and folding under controlledconditions, potentially resulting greater recovery of biologicallyactive product.

From the foregoing it should be readily apparent to one skilled in theart that the ability conferred by the present invention, to transformgenes coding for a protein or an entire metabolic pathway onto achromosome, is extremely useful. Envisioned in this regard, for example,is the application of the present invention to a variety of situationswhere genes from different genetic loci are placed on a chromosome. Theplacement of genes coding for ethanol production is only one example ofthis novel inventive concept. Pursuant to the principles described here,genes coding for alcohol dehydrogenase activity from a variety oforganisms can be combined with genes coding for the pyruvatedecarboxylase activity from a variety of organisms in order to createthe desired pathway. Genes coding for proteins needed for other pathwayscould also be incorporated chromosomally. It should also be apparent toone skilled in the art that, for the ethanolic pathway described here,it is not necessary that the genes coding for alcohol dehydrogenase andpyruvate decarboxylase activities be under common control.

The present invention is further described below with regard to thefollowing illustrative examples.

EXAMPLE 1 Construction of Integration Plasmids Containing E. coliPyruvate Formate-Lyase Gene

The following materials and methods were used throughout the presentexamples unless otherwise stated. E. coli TC4 was used for all geneticconstructions. See Conway, T., Y. A. Osman, J. I. Konnan, E. M. Hoffman,and L. O. Ingram [1987] J. Bacteriol. 169: 949-954. Luria brothcontaining the appropriate selective antibiotic and the indicatedconcentrations of glucose was used in all growth experiments.Antibiotics were used in the following final concentrations except asnoted: ampicillin, 50 μg/ml; chloramphenicol (Cm), 20 μg/ml or 600μg/ml, as indicated; tetracycline, 12.5 μg/ml. ADH indicator platescontaining the Schiff reagent were used to detect aldehyde produced fromethanol by recombinant E. coli expressing ADHII of Z. mobilis. Conway,T., G. W. Sewell, Y. A. Osman, and L. O. Ingram [1987] J. Bacteriol.169: 2591-2597.

Standard procedures were used for plasmid preparation, restrictionenzyme digestions, ligations, transformations, and gel electrophoresis.Isolation of restriction fragments for subsequent cloning wasaccompanied by elution from GTG agarose (FMC BioProducts, ME) usingMicrofilterfuge tubes (Rainin Instrument Co.).

The procedure used for construction of integration plasmids containingan E. coli pyruvate formate-lyase gene (pfl) is shown in FIG. 1. PlasmidpHB4 (Sawers et al. [1988], supra) carrying an incomplete pyruvateformate-lyase (pfl) gene of E. coli was partially digested with BamHI,the 5′-protruding ends were filled in with the Klenow fragment of DNApolymerase I, and the fragments were rejoined by ligating to a SAlIlinker (dCGTCGACG). Those in which the BamHI site outside the pfl genewas replaced by a SalI site were identified by screening of individualtransformants. The resulting plasmid, pLOI513, contained two SalI siteswhich allow removal of the pfl portion of the plasmid from the rest ofthe plasmid.

A chloramphenicol resistance marker gene was excised as an 1.37-kb HhaIfragment from the plasmid cloning vector, pBR325. See Prentki, P., F.Karch, S. Iida, and J. Meyer [1981] Gene 14: 289-299. This HhaI fragmentwas treated with Klenow polymerase to fill protruding ends and ligatedinto the Klenow-treated BamHI site downstream from the adhB gene inpLOI295, see Ingram, L. O., T. Conway, D. P. Clark, G. W. Sewell, and J.F. Preton [1987] Appl. Environ. Microbiol. 53: 2420-2425, in the sameorientation of those of pdc and adhB genes to generate pLOI515. PlasmidpLOI515 was then digested with SalI and partially digested with EcoRI,and, after a Klenow polymerization to generate blunt ends, ligated withBamHI linker (dCCGGATCCGG). The ligation mixture was separated on a GTGagarose gel. The fragment carrying promoter-less pdc and adhB genes andthe Cm^(r) gene (with promoter) was isolated and digested with BamHI.The BamHI fragment in which EcoRI and SalI sites were regenerated oneach end of the original EcoRI-SalI fragment, was cloned in to the BamHIsite in the polylinker of pLOI505 generated by removing SalI site frompUC19 (available from Bethesda Research Laboratories). The SalI siteupstream from the Cm^(r) gene in pLOI506 was removed by digestion withSalI, filling in with Klenow polymerase and religation to generatepLOI508. The 4.6-kb BamHI fragment of pLOI508 was inserted into theBamHI site in the incomplete pfl structural gene carried on plasmidpLOI513. The resultant plasmid pLOI510 was used to introduce the pdc,adhB, and Cm^(r) genes into the chromosome of E. coli strain B (ATCC11303) by homologous recombination as described below (Example 4).

EXAMPLE 2 Construction of Integration Plasmids Containing E. coliPyruvate Formate-Lyase Gene and Temperature-Sensitive for PlasmidReplication

Plasmid pLOI295 was digested with EcoRI and SalI, and the 3.2-kbEcoRI-SalI fragment carrying pdc and adhB genes was treated with Klenowfragment of DNA polymerase to produce blunt ends. This blunt-endedfragment was ligated into the Klenow-treated BamHI site of pLOI513carrying the incomplete pfl gene in the same orientation with respect totranscription to give pLOI542. pLOI542 was then digested with SalI andthe 7.2-kb SalI fragment was ligated to SalI site in the polylinker ofpMAK705, see Hamilton, C. M., M. Aldea, B. K. Washburn, P. Babitzke, andS. R. Kushner [1989] J. Bacteriol. 171: 4617-4622, containing thetemperature-sensitive replicon and Cm^(r) gene (FIG. 2). The resultantplasmid pLOI543 was introduced into the E. coli chromosome by homologousrecombination (see Example 3 below).

EXAMPLE 3 Chromosomal Integration in E. coli B of Z. mobilis pdc andadhB Genes with Loss of an Associated Antibiotic Resistance Gene

The recombinant plasmid pLOI543, which replicates at 30° C., but not at44° C., was used to transform E. coli B with the Cm^(r) gene to give itresistance to chloremphenicol. Transformed cells were grown at 44° C. in100 ml Luria broth containing 20 μg/ml chloramphenicol and 5% (w/v)glucose for 24 hours to select for integration of the plasmid into thechromosome. A portion (0.1 ml) of this culture was diluted, and 0.1 mlof the diluted cell suspension was used to inoculate 100 ml of Luriabroth containing 5% (w/v) glucose without chloramphenicol. The culturewas grown at 30° C. for 12 hours to allow excision. Two or more cyclesof growth were carried out by diluting a portion (0.1 ml) of the cultureand inoculating into 100 ml of fresh medium. A diluted cell suspensionof the 12-hour culture was then used to inoculate Luria broth containing5% (w/v) glucose and incubated at 44° C. to eliminate plasmid. Finally,single colonies were isolated by plating serial dilutions of the cultureonto Luria agar plates containing 2% (w/v) glucose and growing them at30° C. Single colonies appearing on the plates were screened forethanol-production genes by using aldehyde indicator plates and for lossof plasmid as sensitivity to chloramphenicol. Two clones were selectedwhich were sensitive to chloramphenicol but contained theethanol-production genes, strains KO1 and KO2. These clones producedpink colonies on aldehyde indicator plates.

EXAMPLE 4 Chromosomal Integration in E. coli B of Z. mobilis pdc andadhB Genes with Retention of an Associated Antibiotic Resistance Gene

Plasmid pLOI510 was digested with SalI and the 8.6-kb SalI segmentcontaining an incomplete pfl gene was circularized by self-ligation at alow DNA concentration. The covalently closed DNA fragment lackedsequences allowing autonomous replication. E. coli B was transformedwith the ligation mixtures by selection for resistance to 20 μg/mlchloramphenicol. Expression of adhB gene in chloramphenicol resistanttransformants was confirmed by plating colonies from chloramphenicolplates to ADH indicator plates. A single clone was selected for furthercharacterization, designated strain KO3. The lack of plasmid-borne genefor adh or for chloramphenicol resistance, was confirmed by the lack oftransformants from DNA preparations and by direct analysis on agarosegels. This clone produced pink colonies on aldehyde indicator plates.

EXAMPLE 5 Generation and Detection of Mutations that Increase Expressionof Z. mobilis pdc and adhB Genes in the Absence of a Selectable MarkerGene

In Example 3, chromosomal integration in E. coli B of Z. mobilis pdc andadhB genes was carried out under conditions that resulted in loss of theantibiotic resistance gene that was associated with those ethanol geneson the DNA segment used to transform the host cell. Strain KO2,resulting from the procedure of Example 3, was mutagenized with methylmethane sulfonate under standard conditions well known in the art ofbacterial genetics. Surviving mutagenized cells (about 4-8×10⁴) wereplated on aldehyde indicator plates at approximately 200 to 400 coloniesper plate. Four dark red clones indicative of increased ADHII expressionwere isolated and tested for ethanol production as described in Example8 below. Of these mutants, the one with the highest yield in ethanolproduction was designated as strain KO20.

EXAMPLE 6 Selection for Mutations that Increase Expression of Z. mobilispdc and adhB Genes by Selection for Increased Expression of anAssociated Antibiotic Resistance Gene

In Example 4, chromosomal integration in E. coli B of Z. mobilis pdc andadhB genes was carried out under conditions that resulted in retentionof the antibiotic resistance gene that was associated with those ethanolgenes on the DNA segment used to transform the host cell. A series ofdilutions of strain KO3, which resulted from the procedure of Example 4,were plated on Luria agar plates containing 2% glucose and 600 μg/mlchloramphenicol. Large, fat colonies were observed after overnightincubation, at a frequency of approximately 1 per 100,000 plated cells.These large colonies produced ethanol and tested bright red on alcoholindicator plates. Two strains were investigated further, strains KO4 andKO5.

EXAMPLE 7 Insertion of recA and frd Mutations

The recA mutation in E. coli strain JC10240 was transferred to strainsKO4 and KO5 by conjugation with selection for resistance to both Cm andtetracycline (nearby Tn10). Co-inheritance of the recA phenotype wasconfirmed by increased UV sensitivity.

An E. coli Hfr strain capable of mobilizing the frd mutation wasconstructed by transducing the frd deletion mutation from DW12[zid::Tn10, Δ(frdABCD)] into strain KL282 with selection for Tn10. SeeBlaut, M., K. Whittacker, A. Valdovinos, B. A. C. Ackrell, R. P.Gunsalus and G. Cecchini [1989] J. Biol. Chem. 264: 13599-13604. frdmutants were identified among the tetracycline-resistant transductantsby loss of fumarate reductase activity. The resulting Hfr strain,SE1706, was used to conjugate the frd deletion into strain KO4.

Tn10 was deleted from constructs by selection on a modified fusaric acidmedium. See, e.g., Maloy, S. R., and W. D. Nunn [1981] J. Bacteriology145: 1110-1112. This medium contained per liter: 15 g agar, 5 gtryptone, 5 g yeast extract, 20 g glucose, 10 g NaCl, 50 mgchlortetracycline, 10 g NaH₂PO₄, 12 mg fusaric acid, and 10 mM ZnCl₂.Stocks of chlortetracycline (12.5 mg/ml) and fusaric acid (1 mg/ml) wereprepared in 70% ethanol. Complex medium components containingchlortetracycline, sodium phosphate and zinc chloride were autoclavedseparately and mixed after cooling. Antibiotics in 70% ethanol areself-sterilizing. For selection of Tn10 loss, serial dilutions of logphase cultures were spread on fusaric acid plates and incubatedovernight at 37° C. Resulting colonies were streaked for isolation onadditional fusaric acid plates and tested for the loss of tetracyclineresistance. Three strains were constructed KO10 (recA), KO11 (frd), andKO12 (recA, frd).

EXAMPLE 8 Characterization of Ethanol Gene Expression

Fermentation experiments: Fermentations were carried out in Luria brothsupplemented with 10% (w/v) glucose or 8% (w/v) xylose. Fleakers(500-ml, Fisher Scientific Company, Orlando, Fla.) containing 350 ml ofthe medium were equipped with a pH electrode, gas outlet, and samplingport through appropriately drilled rubber caps. A Jenco model 3671 pHcontroller (Whatman Lab Sales, Hillsboro, Oreg.) was used to maintain apH of 6.0 by the addition of base (2 N KOH). Batch fermentations werecarried out in duplicate at 30° C. and were stirred continuously by a1.25 inch star-shaped magnetic bar (100 rpm).

Inocula were grown overnight at 30° C. from isolated colonies inunshaken flasks. Fermentations were inoculated to an initialO.D._(550 nm) of approximately 1.0 (330 mg dry weight of cells/liter).

Cell growth was monitored turbidimetrically at 550 nm using a Bausch andLomb Spectronic 70 spectrophotometer. Ethanol was measured by gas-liquidchromatography as described, for example, by Dombek, K. M., and L. O.Ingram [1986] Appl. Environ. Microbiol. 51: 197-200. Values forconversion efficiency were corrected for fermentation volume changescaused by the addition of base and assumed that all sugar initiallyadded had been metabolized. Volumetric productivities and specificproductivities were estimated during the early stages of fermentation (6to 24 hours) and represent maximum values. All fermentation data intables and in figures represent averages from two or more batchfermentations.

Analysis of volatile and non-volatile acids in the culture: Samples wereremoved for organic acid analyses after 72 hours of fermentation.Volatile and non-volatile acids were measured by gas-liquidchromatography with a Gow-Mac Series 580 gas chromatograph (Gow-MacInstrument Company, Bridgewater, N.J.) connected to a Hewlett-Packard3390A integrator. Nonvolatile acids were converted to methyl estersprior to analysis.

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE):Cells were grown for 24 hours under the conditions of the fermentationexperiments, chilled to 0° C., harvested by centrifugation (7,000×g, 10minutes), washed twice with ⅓ volume of 5 mM sodium phosphate buffer (pH6.5) containing 10 mM 2-mercaptoethanol, and stored frozen at −20° C.Cell pellets were resuspended in an equal volume of buffer and broken bytwo passages through a French pressure cell at 20,000 psi. Membraneswere removed by centrifugation for 90 minutes at 100,000×g. Thesupernatants were separated with a Biorad Mini-Protein IIelectrophoresis unit (Biorad Laboratories, Richmond, Calif.) using an 8%acrylamide denaturing sodium dodecyl sulfate gel. Protein was measuredwith the Bradford reagent. See Bradford, M. M. [1979] Mol. Gen. Genet.181: 548-551. Approximately 20 μg of protein was loaded into each lane.

Enzyme activity: PDC activity was measured in heat-treated French pressextracts as described, for example, by Conway, T., Y. A. Osman, J. I.Konnan, E. M. Hoffmann and L. O. Ingram [1987] J. Bacteriol. 169:949-954. Heat treatment was used to inactivate competing native enzymeswhich complicate measurements of PDC in recombinant E. coli.

EXAMPLE 9 Ethanol Production from Cellobiose, Amorphous Cellulose, andCrystalline Cellulose by Recombinant Klebsiella oxytoca ContainingChromosomally Integrated Z. mobilis Genes for Ethanol Production

Three approaches were used to integrate PET genes into the chromosome ofK. oxytoca M5A1 (Orsdov [1984] p. 461-465, in N. R. Kreig and J. G. Holt(ed.), Bergey's manual of systematic bacteriology, vol. 1. The Williamsand Wilkins Co., Baltimore). Since K. oxytoca and E. coli are closelyrelated, the E. coli pfl gene was used a potential source of homologousDNA to promote recombination analogous to the method used for E. coli(Ohta et al. Appl. Environ. Microbial. 57:893-900). SalI fragments of8.6 kbp were purified from pLOI510 which contained PET genes and catwithin the pfl gene of E. coli. This fragment (lacking genes involved inreplication) was circularized by ligation and transformed into M5A1 toallow direct selection for integration. A shorter 5 kbp PstI fragmentcontaining only a small amount of flanking E. coli pfl was used in asimilar manner. In the third approach, homologous K. oxytoca M5A1 DNA (2kbp random Sau3A fragments) was ligated to a 4.6 kbp BamHI fragmentcontaining only the PET genes with cat (no E. coli DNA) and used fortransformation. Integrated strains were recovered from all threeapproaches by selection on Luria broth plates containing 2% glucose and20 μg Cm/ml. Three integrated clones were recovered with the SalIfragment, two with the PstI fragment and one with the BamHI fragment.

After overnight growth in liquid culture, 0.1 ml of stationary phasecells was spread on plates containing 600 μg Cm/ml and 2% glucose toselect for high level expression. Single large colonies were retainedfrom each independent integration event and named according to therestriction site used for construction, i.e., S1, S2, S3, P1, P2, andB1. These clones were tested for the presence of plasmids bytransformation of miniscreen DNA and for expression of PET genes onaldehyde indicator plates (Table 5). Putative integrated strains foundto contain plasmid-borne cat genes were discarded after digestingminiscreen DNA to confirm the presence of pLOI510 (presumably a lowlevel contaminant of gel-purified fragments). The parent organism andM5A1(pLOI555), an excellent ethanol producer, were included as negativeand positive controls, respectively. Two clones expressed the ethanolgenes at levels nearly equivalent to that of that of M5A1(pLOI555),strains B1 and P2.Plasmid LOI510, containing PET genes and cat withinthe E. coli pfl gene was digested with SalI, or Pst1, circularized andtransformed into K. oxytoca strain M501. See Wood et al., Appl. Environ.Microbiol. 58:2103 (1992), which is herein incorporated by reference.Additionally, homologous K. oxytoca M5A1 DNA prepared by Sau3A digestionwas ligated to a 4.6 kb BamH1 fragment containing only the PET and catgenes was also used for transformation. Recombinants were initiallyselected using 20 μg chloramphenicol/ml and expressed low levels of Z.mobilis enzymes. As with E. coli (Ohta et al. [1991] Appl. Environ.Microbiol. 57: 893-900), expression was boosted by direct selection ofmutants with resistance to 600 μg chloramphenicol/ml. A single cloneexpressing high level resistance was retained for each independentintegration event.

Fermentations were carried out in 500-ml Fleakers which served as pHstats (350 ml working volume) essentially as described previously (Beallet al., Biotechnol. & Bioeng. 38: 296-303 (1991)). Luria brothcontaining either 10% glucose or 10% cellobiose was tested at 30° C., pH6.0, 100 rpm. Inocula for fermentations were grown at 30° C. overnightin unshaken, 250-ml flasks containing 50 ml of Luria broth (4% glucose).After mixing, cell densities were measured at 550 nm and used tocalculate the volume required to provide an initial density of 0.32 mgcell dry weight/ml (O.D. 550 nm of 1.0). Cells were harvested from thisvolume by centrifugation and resuspended in a portion of the broth fromeach respective ph-stat to start fermentation.

Sugars were sterilized separately by filtration. Cellulose fermentationscontained 50 g/liter SOLKA FLOC SW40 (James River Corporation, SaddleBrook, N.J.) and were carried out at 35° C. Cellulose was sterilized byautoclaving as a dry powder. For investigations of cellulosefermentation using commercial enzymes, CYTOLASE or MULTIFECT, was addedat the time of inoculation.

Samples were removed for the determination of cell mass (O.D. 550 nm)and ethanol (gas liquid chromatography; Beall et al., supra). Ethanolconcentrations are expressed as g/liter. Ethanol yields were correctedfor dilution by the addition of base during fermentation and computed onthe basis of total sugar or cellulose initially present. No correctionswere made for residual carbohydrates. Maximum theoretical yields fromglycolysis and fermentation are 0.51 g ethanol/g glucose, 0.536 gethanol/g cellobiose, and 0.56 g ethanol/g cellulose. Except asindicated, results presented are an average of 2 or more fermentations.TABLE 5 Fermentation of 10% glucose (48 hours) by strains of M5A1containing integrated pet genes. Base Cell Mass^(b) Ethanol Aldehyde^(c)Strain^(a) (mM/liter) (g/liter) (g/liter) Plates Plasmid^(d) M5A1 1083.2 15 − − M5A1 91 5.1 45 ++++ + (pLOI555) S1 120 2.9 37 + − S2 171 3.244 ++ − S3 97 2.4 37 ++ − P1 120 2.0 38 ++ − P2 91 3.7 44 +++ − B1 634.0 47 ++++ −^(a)Values reported are from a single experiment although many werereproduced and averaged in subsequent investigations.^(b)Cell mass was calculated from the maximal O.D. at 550 nm (approx.0.32 g dry weight/liter per O.D. unit).^(c)Comparative rate of color development on aldehyde indicator platesat 30° C. was used as a relative measure of expression from the petoperon.^(d)Small scale preparations of DNA were examined on agarose submarinegels and tested for their ability to transfer antibiotic resistance toE. coli DH5α during transformation.

Comparison of glucose fermentation by integrated strains to5A1(pLOI555): After 48 hours, high levels of ethanol equivalent toM5A1(pLOI555) were produced by 4 strains containing PET integrations(Table 5). The two which produced the lowest levels of acidicco-products also grew to the highest densities. These strains, P2 andB1, were selected for further investigation.

Fermentation of glucose and cellobiose by strains P2 and B1. Strains P2and B1 were investigated for their ability to ferment 10% glucose and10% cellobiose (Table 6). During glucose fermentation (FIG. 5), bothintegrated strains produced 3 times more ethanol than the parentorganism, M5A1, but were slightly inferior to M5A1(pLOI555)(plasmid-borne PET genes) in yield, and in volumetric productivity.Unexpectedly, integration and high level expression of PET genes instrain B1 was accompanied by a loss of ability to ferment cellobiose.Strain P2, however, grew well and produced ethanol from cellobiose (FIG.6) with 96% of the maximum theoretical yield.

FIGS. 5 and 6 thus illustrate the ethanol production by recombinantstrains of K. oxytoca M5A1. FIG. 5 illustrates the production fromglucose (100 g/liter). Symbols: ●, strain M5A1(pLOI555); ▴, strain P2containing integrated PET genes; ▪, strain B1 containing integrated PETgenes; ◯, M5A1 control. FIG. 6 illustrates the production fromcellobiose (100 g/liter) fermentation by strain P2. Symbols: ▴, ethanol;Δ, cell mass.

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and the scope of the appended claims. TABLE 6 Ethanolproduction by recombinant strains of K. oxytoca M5A1. Volumetric Max. %Productivity EtOH Yield Theoretical^(e) Organism^(a) SubstrateAdditives^(b) (g/liter/h) (g/liter) (g/g S) Yield M5Al 10% glucose none0.72 16.2 0.17 33 M5Al(pLOI555) ″ ″ 2.1 48.0 0.50 99 P2 ″ 1.6 46.4 0.4690 B1 ″ ″ 1.5 47.6 0.49 96 P2 10% cellobiose ″ 1.5 45.2 0.49 96 B1 ″ ″0.1 1.2 0.01 2 P2 none ″ 0.04 1.0 — — P2 none  10% CYTOLASE 0.12 4.7 — —P2 none  10% MULTIFECT 0.06 5.2 — — P2 5% SOLKA FLOC SW40 no enzyme 0.052.0 0.04 8 P2 5% SOLKA FLOC SW40 0.1% CYTOLASE 0.08 2.3 0.07 12 P2 ″0.5% CYTOLASE 0.13 7.0 0.14 26 P2 ″ 1.0% CYTOLASE 0.18 8.7 0.18 32 P2 ″5.0% CYTOLASE 0.52 16.9 0.36 64 P2 ″  10% CYTOLASE 0.50 16.3 0.35 62 P25% SOLKA FLOC SW40 0.1% MULTIFECT 0.08 3.3 0.07 12 P2 ″ 0.5% MULTIFECT0.18 5.9 0.13 23 P2 ″ 1.0% MULTIFECT 0.36 10.4 0.23 41 P2 ″ 5.0%MULTIFECT 0.76 23.8 0.51 92 P2 ″  10% MULTIFECT 0.86 32.5 0.69 123^(a)Glucose and cellobiose fermentations were carried out at 30° C.;other fermentations were carried out at 35° C.^(b)Enzymes were added at the time of inoculation.^(c)Computed for the time period between 6 and 24 hours.^(d)Corrected for dilution by the addition of base during fermentation.S (substrate) refers to glucose, cellobiose or SOLKA FLOC SW40.^(e)Based on a maximum theoretical yield of 51 g ethanol/100 g glucose,53.5 g ethanol/100 g cellobiose, and 28 g ethanol/50 g cellulose. Valueswere not corrected for residual substrate.

1-16. (canceled)
 17. A process for producing a recombinant host cellstrain that produces high levels of a desired polypeptide, comprisingthe steps of: (a) providing a culture comprised of enteric bacterialhost cells comprising a pyruvate formate lyase promoter whichis-endogenous to said host cells and a DNA encoding a pyruvate formatelyase gene under transcriptional control of said promoter; (b)transforming host cells in said culture with a heterologous DNA moleculecomprising (i) two genetic elements assembled such that the codingregions of both elements are translated in the same direction, whereinthe downstream genetic element comprises a selectable marker gene, apromoter that controls the transcription of said selectable marker gene,and a transcription termination sequence, and wherein the upstreamgenetic element comprises one or more promoterless coding regionsencoding at least one desired polypeptide followed by a transcriptiontermination sequence, and (ii) sequences that flank said geneticelements and are oriented such that their direction of translation isthe same as that of the two heterologous genetic elements, and (iii)sequences that flank said genetic elements and are sufficientlyhomologous to said pyruvate formate lyase gene to enable integration byhomologous recombination, whereby integration of said genetic elementsinto said pyruvate formate lyase gene results by means of homologousrecombination; (c) selecting for host cells produced in step (b) thatexpress said selectable marker polypeptide at a first level; (d)screening host cells obtained in step (c) to obtain host cells thatproduce said desired polypeptide at an initial level; (e) optionallyexposing host cells identified in step (d) to a mutagen under conditionssuch that mutations are created in said DNA; and then (f) testing hostcells produced in step (d) or step (e) for host cells that produce saidmarker polypeptide at a level higher than said initial level, to obtainhost cells having a mutation that causes increased expression of theupstream genetic element resulting in an increase in production by saidhost cells of all polypeptides encoded by said heterologous DNA moleculecompared to said production of all polypeptides encoded by saidheterologous DNA molecule by said host cells in the absence of saidmutation, wherein said increased expression is retained in the absenceof conditions that select for cells having said increased expression.18. The process according to claim 17, wherein said strain is a strainof Escherichia coli and wherein further (i) in step (b) said DNAmolecule is a plasmid, wherein said plasmid comprises a replicon that istemperature-sensitive for replication; (ii) in step (b) saidtransforming host cells further comprises introducing said plasmid intosaid host-cells and growing said host cells under conditions that selectfor cells that express said selectable marker gene at said first leveland at a temperature that does not permit replication of said plasmid,resulting in integration of said plasmid into said host gene of saidchromosome by homologous recombination; and (iii) in step (c) saidselecting for host cells further comprises (1) growing said host cellsthat express said selectable marker gene, resulting in excision fromsaid host gene of said temperature-sensitive replicon and of saidplasmid, and wherein further (2) said host cells are grown under saidconditions at a second temperature that does not permit replication ofsaid plasmid, resulting in host cells that retain said heterologous DNAmolecule encoding said desired polypeptide in the absence of saidplasmid.
 19. A process according to claim 17, wherein further (i) instep (b) said DNA molecule comprises a closed circular DNA lacking anability to replicate, and (ii) in step (f) said testing host cellscomprises selecting for host cells produced in step (d) or step (e) thatexpress said selectable marker gene At a second level that is higherthan said first level, and then screening said host cells that expresssaid selectable marker gene at said second level for host cells thatproduce said desired protein at a level higher than said initial level.20. A process according to claim 17, wherein said selectable markerprotein confers resistance to chloramphenicol on said host cell strain.21. A process according to claim 20, wherein said first level ofexpression of said selectable marker gene confers resistance to at leastabout 20 Ag/ml of chloramphenicol.
 22. A process according to claim 20,wherein said first level of expression of said selectable marker geneconfers resistance to at least about 20/.tg/ml of chloramphenicol andsaid second level of expression of said selectable marker proteinconfers resistance to at least about 100 μg/ml of chloramphenicol.
 23. Aprocess according to claim 20, wherein said coding region of theupstream genetic element of said heterologous DNA segment furthercomprises a second coding region encoding a second desired polypeptide.24. A cell strain according to claim 17, wherein said enteric bacterialhost cell is selected from the group consisting of Erwinia, Escherichiaand Klebsiella.
 25. A process according to claim 24, wherein saidenteric bacterial host cell strain is a strain of Escherichia coli. 26.A process according to claim 23, wherein said coding region for thefirst genetic element of said heterologous DNA molecule encodes analcohol dehydrogenase and a pyruvate decarboxylase.
 27. A processaccording to claim 26, wherein said alcohol dehydrogenase and saidpyruvate decarboxylase are encoded by genes from Zymomonas mobilis. 28.A process according to claim 27, wherein said strain is an Escherichiacoli strain, and said strain is able to produce ethanol by fermentationof glucose or xylose with a theoretical yield corresponding toconversion of at least about 90% of added sugar to ethanol.
 29. Aprocess according to claim 28, wherein said chromosome further comprisesa mutation that impairs succinate production.
 30. A process according toclaim 29, wherein said mutation that impairs succinate productioncomprises a mutation in a fumarate reductase (frd) gene.
 31. A processaccording to claim 25, wherein said chromosome further comprises amutation that impairs recombination in said host cell strain.
 32. Aprocess according to claim 31, wherein said mutation that impairsrecombination comprises a mutation in a recA gene. 33.-39. (canceled)40. A process according to claim 27, wherein said cell is able toproduce ethanol by fermentation of glucose or xylose with a theoreticalyield corresponding to conversion of at least about 90% of added sugarto ethanol.
 41. (canceled)
 42. A method according to claim 26, whereinsaid cell strain is designated K04 and ATCC #55123.
 43. A methodaccording to claim 26, wherein said cell strain is designated K011 andATCC* #55124.
 44. A method according to claim 26, wherein said cellstrain is designated K012 and ATCC #55125.
 45. A method according toclaim 26, wherein said cell strain is designated K020 and ATCC #55126.46. (canceled)
 47. A process according to claim 27, wherein said strainis a Klebsiella oxytoca strain, and said strain is able to produceethanol by fermentation of glucose or cellobiose with a theoreticalyield corresponding to conversion of at least about 90% of added sugarto ethanol. 48.-49. (canceled)